Exploiting metabolic vulnerabilities in breast cancers with NF1 loss
Authors: Yoon-Sim Yap, Jiancheng Hu
Journal: Cell Reports Medicine (2023)

Auf der Maur et al.1Auf der Maur P. Trefny M.P. Baumann Z. Vulin M. Correia A.L. Diepenbruck M. Kramer N. Volkmann K. Preca B.-T. Ramos P. et al.N-acetylcystein overcomes NF1-loss-driven resistance to PI3Ka inhibition in breast cancer.Cell Rep. Med. 2023; 4: 101002Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar identify neurofibromin 1 (NF1) loss as a mechanism of resistance to PI3K inhibitor in breast cancer cells. NF1 loss leads to enhanced glycolysis, which may be targeted with the antioxidant N-acetyl cysteine (NAC). Auf der Maur et al.1Auf der Maur P. Trefny M.P. Baumann Z. Vulin M. Correia A.L. Diepenbruck M. Kramer N. Volkmann K. Preca B.-T. Ramos P. et al.N-acetylcystein overcomes NF1-loss-driven resistance to PI3Ka inhibition in breast cancer.Cell Rep. Med. 2023; 4: 101002Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar identify neurofibromin 1 (NF1) loss as a mechanism of resistance to PI3K inhibitor in breast cancer cells. NF1 loss leads to enhanced glycolysis, which may be targeted with the antioxidant N-acetyl cysteine (NAC). Alpelisib with fulvestrant is one of the preferred treatment options in PIK3CA-mutated advanced breast cancer (BC), after progression on cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) with endocrine therapy (ET). However, PIK3CA-mutated advanced BCs have worse outcomes compared to wildtype tumors,2Mosele F. Stefanovska B. Lusque A. Tran Dien A. Garberis I. Droin N. Le Tourneau C. Sablin M.P. Lacroix L. Enrico D. et al.Outcome and molecular landscape of patients with PIK3CA-mutated metastatic breast cancer.Ann. Oncol. 2020; 31: 377-386Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar with median progression-free survival in the range of 6 months on alpelisib with ET after prior CDK4/6i. Apart from inactivating PTEN mutations, adaptive rewiring, epigenetic and metabolic reprogramming in cancer cells have been implicated in resistance to PI3K inhibition.3Vasan N. Cantley L.C. At a crossroads: how to translate the roles of PI3K in oncogenic and metabolic signalling into improvements in cancer therapy.Nat. Rev. Clin. Oncol. 2022; 19: 471-485Crossref PubMed Scopus (28) Google Scholar To elucidate other mechanisms of resistance to PI3K inhibition, Auf der Maur et al. performed an in vivo genome-wide PiggyBac transposon-mediated mutagenesis screen in parallel with an in vivo resistance screen in a PIK3CAH1047R-mutated murine tumor model, and identified NF1 as a candidate gene driving resistance to PI3K inhibition.1Auf der Maur P. Trefny M.P. Baumann Z. Vulin M. Correia A.L. Diepenbruck M. Kramer N. Volkmann K. Preca B.-T. Ramos P. et al.N-acetylcystein overcomes NF1-loss-driven resistance to PI3Ka inhibition in breast cancer.Cell Rep. Med. 2023; 4: 101002Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar NF1 mutations are enriched in metastatic or post-progression BC specimens (range 5%–18%), based largely on biopsies after progression on ET +/- CDK4/6i.4Razavi P. Chang M.T. Xu G. Bandlamudi C. Ross D.S. Vasan N. Cai Y. Bielski C.M. Donoghue M.T.A. Jonsson P. et al.The genomic landscape of endocrine-Resistant advanced breast cancers.Cancer Cell. 2018; 34: 427-438.e6Abstract Full Text Full Text PDF PubMed Scopus (488) Google Scholar,5Zheng Z.Y. Anurag M. Lei J.T. Cao J. Singh P. Peng J. Kennedy H. Nguyen N.C. Chen Y. Lavere P. et al.Neurofibromin is an Estrogen Receptor-α Transcriptional Co-repressor in breast cancer.Cancer Cell. 2020; 37: 387-402.e7Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar The role of NF1 in tumor suppression beyond regulation of the Ras-Raf-MEK and Ras-PI3K-mTOR cellular proliferation pathways5Zheng Z.Y. Anurag M. Lei J.T. Cao J. Singh P. Peng J. Kennedy H. Nguyen N.C. Chen Y. Lavere P. et al.Neurofibromin is an Estrogen Receptor-α Transcriptional Co-repressor in breast cancer.Cancer Cell. 2020; 37: 387-402.e7Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar,6Yap Y.S. McPherson J.R. Ong C.K. Rozen S.G. Teh B.T. Lee A.S.G. Callen D.F. The NF1 gene revisited - from bench to bedside.Oncotarget. 2014; 5: 5873-5892Crossref PubMed Scopus (110) Google Scholar is increasingly recognised (Figure 1). Transcriptomic analysis by Auf der Maur et al. revealed upregulation of not only MAPK activity, but also increased MYC signaling with knockout (KO) of NF1 in the T47D cells harboring PIK3CA H1047R mutation.1Auf der Maur P. Trefny M.P. Baumann Z. Vulin M. Correia A.L. Diepenbruck M. Kramer N. Volkmann K. Preca B.-T. Ramos P. et al.N-acetylcystein overcomes NF1-loss-driven resistance to PI3Ka inhibition in breast cancer.Cell Rep. Med. 2023; 4: 101002Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar Cancer cells have different metabolic features from normal cells. While both MAPK and MYC signaling are known to enhance aerobic glycolysis via the Warburg effect, and MYC with augmented oxidative phosphorylation to increase cell energy production, the impact of NF1 loss on glycolysis and mitochondrial respiration was previously unclear. Interestingly, although NF1 loss led to enhanced glycolysis with increased lactate production, cellular reactive oxygen species (ROS) levels were reduced with lower mitochondrial respiration, indicating less dependency on oxidative phosphorylation.1Auf der Maur P. Trefny M.P. Baumann Z. Vulin M. Correia A.L. Diepenbruck M. Kramer N. Volkmann K. Preca B.-T. Ramos P. et al.N-acetylcystein overcomes NF1-loss-driven resistance to PI3Ka inhibition in breast cancer.Cell Rep. Med. 2023; 4: 101002Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar Antioxidants suppress oxidation and may augment proliferation of cancer cells by reducing the generation of ROS. Conversely, antioxidants may have anti-cancer properties because ROS may promote tumor migration and invasion. NAC, an antioxidant, was previously shown in preclinical studies to stimulate proliferation and metastasis in melanoma and lung cancer.9Le Gal K. Ibrahim M.X. Wiel C. Sayin V.I. Akula M.K. Karlsson C. Dalin M.G. Akyurek L.M. Lindahl P. Nilsson J. Bergo M.O. Antioxidants can increase melanoma metastasis in mice.Sci. Transl. Med. 2015; 7: 308re308Crossref Scopus (457) Google Scholar NAC was added to alpelisib by Auf der Maur et al. with the expectation of increasing resistance to PI3K inhibition, but intriguingly NAC sensitized NF1 KO cells to alpelisib in vitro, in vivo, and ex vivo in patient-derived organoids harboring PIK3CA H1047R mutation. No additive effect of NAC was seen in T47D control tumors in vivo, which remained highly sensitive to alpelisib. To evaluate the dependency of NF1 KO cells on MYC, KJ-Pyr-9 was used to inhibit MYC pharmacologically. The increased sensitivity of NF1 KO cells to KJ-Pyr-9 compared to control cells supported the crosstalk between NF1 loss and MYC. However, the combination of NAC and KJ-Pyr-9 did not increase anti-proliferative effects. Only NAC, not KJ-Pyr-9, could reverse the increased glycolysis seen in NF1 KO cells. This suggests that the increased glycolysis with NF1 KO is independent of MYC. Notably, binimetinib (MEK inhibitor) showed only modest activity even when combined with alpelisib. What is the mechanistic basis of NAC circumventing resistance to PI3K inhibitor with NF1 loss? Global phosphoproteomic analysis demonstrated further reduction in mTOR signaling with addition of NAC to alpelisib in NF1 KO cells. Given that mTOR signaling is a key driver of glycolysis, it is likely effected through mTOR inhibition. Combining everolimus (mTORC1 inhibitor) with NAC was more effective than either drug alone in diminishing cellular proliferation. Double vertical blockade of the PI3K-Akt-mTOR pathway using everolimus with alpelisib demonstrated similar effectiveness to NAC-alpelisib combination. Is the antitumor effect of NAC restricted to (1) NF1 loss in (2) PIK3CAH1047R-mutated cells treated with (3) alpelisib? To address these questions, the authors silenced NF1 in MCF7 cells that harbor PIK3CAE545K mutation and found similar efficacy with the addition of NAC to alpelisib. Importantly, the potential activity of NAC does not appear to be unique to NF1 loss. Similar activity was observed in T47D cells engineered to overexpress the constitutively active KRAS 4BG12V mutation, supporting the efficacy of NAC co-treatment in tumors harboring other MAPK pathway alterations. Moreover, NAC co-treatment with ribociclib in NF1 KO T47D cells overcame the resistance to CDK4/6 inhibition, while the effect with fulvestrant was marginal. Several questions remain, including the prevalence of NF1 alterations after exposure to PI3K inhibitors, the biological differences underlying the conflicting results with antioxidants in preclinical and clinical studies in various tumors, the complexity of redox homeostasis, and how to design new therapeutics to target these metabolic vulnerabilities in addition to repurposing old drugs. Recently, another study reported that tumor growth of neurofibromin-deficient cells was driven by decreased respiration in an ERK-dependent fashion. Tumorigenicity was hampered by NAD+ or NAD+ deacetylase SIRT3, with synergistic effect from ablation of TRAP1 mitochondrial chaperone.7Masgras I. Cannino G. Ciscato F. Sanchez-Martin C. Darvishi F.B. Scantamburlo F. Pizzi M. Menga A. Fregona D. Castegna A. Rasola A. Tumor growth of neurofibromin-deficient cells is driven by decreased respiration and hampered by NAD(+) and SIRT3.Cell Death Differ. 2022; 29: 1996-2008Crossref PubMed Scopus (6) Google Scholar In spite of such discoveries, metabolic dysregulation, one of the hallmarks of cancer, remains poorly understood. There is a pressing need for comprehensive characterization of the metabolome in human tumors beyond the glycolytic pathway. The interplay with various genomic aberrations, epigenetic remodeling, the tumor microenvironment, and the microbiome needs to be unraveled further. In summary, NAC has promising efficacy based on this preclinical study. It is affordable and may be better tolerated than everolimus to overcome resistance to PI3K or CDK4/6 inhibition. However, the metabolic milieu and tumor microenvironment are more complex in humans than in the laboratory, with considerable heterogeneity and plasticity in response to any perturbation. The optimal dose of NAC required to revert the glycolytic phenotype is also unclear. The development of metabolic strategies to treat cancers in humans has been challenging, with disappointing results from the metformin trials,10Lord S.R. Harris A.L. Is it still worth pursuing the repurposing of metformin as a cancer therapeutic?.Br. J. Cancer. 2023; 128: 958-966Crossref PubMed Scopus (7) Google Scholar though asparaginase in acute lymphoblastic leukemia is a successful example of targeting metabolic vulnerability in cancer cells.8DeBerardinis R.J. Chandel N.S. Fundamentals of cancer metabolism.Sci. Adv. 2016; 2e1600200Crossref PubMed Scopus (1665) Google Scholar To test the hypothesis generated by Auf der Maur et al., the next step would be to conduct a clinical trial in the right patients with a multi-omics approach, and translate the important findings of this study from bench to bedside. Y.S.Y. is supported by Singapore National Medical Research Council (NMRC) Clinician Scientist Award (MOH-CSAINV18nov-0009). Y.S.Y. reports honoraria/personal fees from Novartis, Pfizer, Lilly/DKSH, Astra Zeneca, Eisai, MSD, Specialised Therapeutics, and Roche; travel support from DKSH and Astra Zeneca; and research support from MSD, outside the submitted work. N-acetylcysteine overcomes NF1 loss-driven resistance to PI3Kα inhibition in breast cancerAuf der Maur et al.Cell Reports MedicineApril 11, 2023In BriefAuf der Maur et al. discover a mechanism of resistance to PI3Kα inhibition mediated by loss of NF1 using a genome-wide genetic screen in a breast cancel model. Treatment with the antioxidant N-acetyl cysteine reverts the glycolytic phenotype of NF1 KO cells and sensitizes them to PI3Kα inhibition. Full-Text PDF Open Access